CA3000010A1 - Sorting biological and non-biological moieties using magnetic levitation - Google Patents
Sorting biological and non-biological moieties using magnetic levitation Download PDFInfo
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- CA3000010A1 CA3000010A1 CA3000010A CA3000010A CA3000010A1 CA 3000010 A1 CA3000010 A1 CA 3000010A1 CA 3000010 A CA3000010 A CA 3000010A CA 3000010 A CA3000010 A CA 3000010A CA 3000010 A1 CA3000010 A1 CA 3000010A1
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Abstract
Description
LEVITATION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62/236,692 entitled "Sorting Biological and Non-Biological Moieties Using Magnetic Levitation" filed October 2, 2015, the contents of which are incorporated by reference herein in its entirety for all purposes.
STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
BACKGROUND
These events include cell-cycle stage, differentiation, cell-death (apoptosis / necrosis), malignancy, disease state, activation, phagocytosis, in vivo and ex vivo cell aging, viral infection, and specific as well as non-specific responses to drugs.
SUMMARY OF THE INVENTION
Systems such as these may also be used, for example, as a microfluidic platform for label-free, high throughput isolation of circulating tumor cells (CTCs) from whole blood. The platform employs the principle of magnetic levitation to separate cells based on their density profiles while they flow in a microchannel. Cancer cells that typically have lower intrinsic densities than blood cells may be levitated at a higher level within the microchannel, in which three different paramagnetic fluids flow top-on-bottom (top and middle flows:
carrier buffer, bottom flow: sample blood containing cancer cells). Those levitated cancer cells may then be extracted from the sample blood flow, and are collected within a carrier buffer fluid flowing on the top. Accordingly, the platform enables, for example, the high throughput isolation of rare CTCs from cancer patient's blood, which facilitates clinical studies for CTC-derived biomarkers and molecular targets.
The microcapillary or microfluidic channel is defined by a plurality of layers having portions of the microcapillary or microfluidic channel formed therein with at least one of the plurality of layers providing an inlet channel into the microcapillary or microfluidic channel and at least two of the plurality of layers providing separate outlets from the microcapillary or microfluidic channel.
In other forms, the magnetic field may be generated by coils induced by electrical current.
Still other forms of magnetic field generation may also be used.
The system includes a levitation device for separating a heterogeneous population of cells in which the variances in the cells are based on differences in magnetic susceptibility.
The device includes at least one magnet producing a magnetic field in which the magnet(s) is/are proximate a microcapillary or microfluidic channel for reception of a sample containing a heterogeneous population of cells. The system further includes a light source, a lens (in some forms of the system, although the lens may be omitted), and a frame. The frame supports the levitation device, the light source, and, if present as part of the system, the lens. This frame is further configured to support an imaging device. The frame supports the light source in a position to transmit light through the levitation device and, if present, the lens to the imaging device for real-time observation of at least a portion of heterogeneous population of cells.
camera, and a CCD camera. The data collected by the imaging device may be transmitted over Bluetooth or wifi or phone networks, particularly when the site of operation is the a remote point of care.
When the frame is in the expanded position, the frame may be configured to receive the imaging device in recesses on a top surface thereof.
The system further includes at least one needle at the inlet and/or outlet of the microcapillary or microfluidic channel for introducing or withdrawing fluid from the microcapillary or microfluidic channel at a respective pre-defined position over the height and/or width of the microcapillary or microfluidic channel.
BRIEF DESCRIPTION OF THE DRAWINGS
1C illustrates the i-LEV set-up including a smartphone, lens, levitation device, light source, and filters supported by a surrounding frame.
platform. FIG. 2A shows WBC and RBC separation image taken by i-LEV. FIG. 2B
shows RBC
and WBCs levitated at different heights are imaged by conventional microscopy using bright field. FIG. 2C shows fluorescent images of CD45-labeled WBC. FIG. 2D
shows the overlap of the bright field and CD45 images to confirm the separation of WBC
and RBC.
FIG. 2E shows live-dead assay imaging of RBCs and WBCs by i-LEV in which live RBCs levitate while dead WBCs aggregate at bottom of the capillaries. FIG. 2F shows bright field, FIG. 2G shows DAPI-labeled, and FIG. 2H show overlapped images of WBC using fluorescence microscopy.
concentrations varying from 250 million cells/mL to 0.8 million cells/mL and FIG. 3D plots the width of the blood band for each of these RBC concentrations with the graph being linear within a cell concentration range between 50 and 250 million cells/mL.
FIG. 3E
includes images of levitated WBC at different concentrations and FIG. 3F plots the width of the blood band is against the WBC concentration.
FIG. 4A is an image of RBC at a concentration of 100,000 cells/mL and FIG. 4B illustrates the detection of single blood cells using image algorithms. FIG. 4C illustrates density measurement of polyethylene beads in the magnetic levitation platform in which beads between 10-100 um in diameter with different densities (1.025 g mL-1, 1.031 g mL-1, 1.044 g mL-1, and 1.064 g mL-1) are shown to have distinct levitation heights in 30 mM Gd+. FIG.
4D illustrates beads with 1.064 g mL-1 density have different levitation heights at different Gd+ concentrations (10 mM, 30 mM, 60 mM). FIG. 4E shows a linear fitting curve that provides a standard function to measure densities of particles.
concentrations.
FIGS. 5A-5C are fluorescence microscopy images of WBC and RBC separation in different Gd+ concentrations (30, 60 and 90 mM), while FIGS. 5D-5F are the same process imaged using the i-LEV system. Both imaging platforms show that rising Gd+
concentrations increase levitation height, whereas separation resolution decreases.
FIG. 7A shows that the levitation of red blood cells over time that 75 mg/mL of chloroquine was spiked into.
FIG. 7B shows the levitation heights and image analysis of chloroquine concentrations of 1.25, 2.5, 5 ad 7.5 mg/mL. FIG. 7C is a zoomed in graph of FIG. 7B showing a more detailed graph to show effects of each concentration. FIG. 7D are the actual images of analyzed to generate the data sets in FIG. 7B and 7C in which density beads were added each time for calibration experiments.
The mobile application reports the measurements to the healthcare provider.
The healthcare provider analyzes the results and provides feedback through an online system.
Ga3+ (FIG. 24A) and 100 mM Ga3+ (FIG. 24B) using the device of FIG. 23.
cells have significantly different levitation profiles in the magnetic levitation-based platform.
DETAILED DESCRIPTION OF THE INVENTION
In each of the sub-sections below various aspects are described including the construction of an "i-LEV"
device, which is a portable levitation system which may incorporate an imagining device (such as a smartphone) to provide point of care (POC) imaging and analysis of a sample containing a population of moieties. Subsequent description of moiety sorting and blocking techniques are then further described. Finally, the use of such levitation systems in the construction of 3-D self-assembled systems are described.
Integrating cell phone imaging with magnetic levitation (i-LEV)
Using this portable imaging magnetic levitation system (an example of which, the i-LEV, being referred to herein by name), it is shown that white and red blood cells can be identified and cell numbers can be quantified without using any labels. In addition, cells levitated in i-LEV can be distinguished at single cell resolution, potentially enabling diagnosis and monitoring, as well as clinical and research applications.
Moreover, POC
devices can be applied to monitor compliance and disease progression. However, most systems require extensive sample preparation and labeling steps, which limit their usage.
Precision medicine tailors treatments to a patient's profile based on their genetic data.
Cellular and molecular analyses are increasingly being performed by research institutions and drug companies to achieve more efficient drug development and improved early diagnoses. In this respect, smartphones with high-resolution cameras, fast computing power, graphics processors, data storage and connectivity capacities are used for various healthcare platforms, including telemedicine and POC diagnostics.
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Coulter and flowcytometry are complex and expensive, whereas hemocytometry is inexpensive but labor intensive, time-consuming and not practical for POC
testing. Recently developed methods have advanced the field by applying sensitive and robust technologies.
However, an inexpensive and accurate blood count analyzer is still missing for POC
treatment.
The i-LEV
device is an easy-to-use and easy-to-access POC solution for blood cell counting that could be used to monitor disease progression and drug effectiveness in the home-setting.
Example I: i-LEV construction and design
system can accommodate fluorescent imaging hardware by inserting broadband LEDs, as well as excitation and emission filters. Micro capillary channel (1 mm x 1 mm cross-section, 50 mm length and 0.2 mm wall thickness), N52 grade neodymium magnets (NdFeB) (50 mm length, 2 mm width and 5 mm height), and side mirrors are used to build the magnetic levitation device as illustrated in FIG. 1B.
and 90 mM Gd+). 30 uL of sample were pipetted into the micro capillaries and the channel was sealed with CritosealTM. The samples were levitated for 30 min until they reached their equilibrium height within the system. Calibration measurements were performed to quantify stabilization time as shown in FIG. 3A and 3B. The width and height of the cells and beads were imaged and analyzed using image).
pellet was re-suspended in PBS. Incremental concentrations between 1 and 5 million WBC/mL
were used to correlate the width of the WBC levitation band with the cell concentrations.
were then washed twice with PBS and re-suspended in PBS. At the end of this process, live WBC
and lx RBC were suspended (50:50) in PBS with 30 mM Gd+ at 1,500 rpm for 3 min. Cells were levitated for 30 min and imaged.
lysis, WBC were frozen overnight at -80 C in PBS without a cryoprotective agent in order to kill WBC. After overnight incubation, dead WBC cells were stained with 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) (1:1,000 Invitrogen) for 15 min at room temperature.
After staining, dead WBC were washed twice with PBS and re-suspended in PBS.
Finally dead WBC and 1.000x RBC were mixed and suspended (50:50) in PBS with 30 mM Gd+
at 1,500 rpm for 3 min. Cells were levitated for 30 min and imaged.
was performed using Image). Briefly, the image taken by the smartphone was uploaded to Image). Then, the levitated blood band was cropped and the background was subtracted.
The image was converted to 16-bit and the threshold was adjusted to "Default-BW"
settings. Area, center of mass, and bounding rectangle were measured. Dividing the measured area by the bounding rectangle provided the average height of the blood band.
Each step of image analysis is explained in more detail in the Supplementary Information.
additional components such as simple LED light sources and ND filters to improve the images. The front panel has a slide-in door to block external light. The set-up was designed using poly-methyl methacrylate (PMMA) building blocks prepared with a laser cutter. The levitation device is made of magnets, mirrors, and channel. Two permanent magnets (50 mm length, 2 mm width and 5 mm height) are set up in such an orientation that the same poles face each other. A capillary channel (50 mm length, 1 mm x 1 mm cross-section, 0.2 mm wall thickness) can be inserted between the magnets. Side mirrors are used to illuminate and observe the levitation channel. Samples spiked into a paramagnetic medium (in this instance, Gadavist) are levitated inside the medium at a position where the buoyancy force and the magnetic force are equal. The levitation height of the sample is calculated based on Equation 1.
¨Ax B.VB ¨ Apg = 0 (Eq. 1) Ito The first part of the equation represents the magnetic force applied to a particle, while the second part represents the buoyancy force. The magnetic induction, gravitational acceleration, difference between volumetric densities of cells and medium, and permeability of the free space are represented by B, g, p, and /Jo, respectively. Samples are levitated at unique heights mainly based on their density, independent of their mass and volume. To demonstrate the i-LEV system's potential to separate different sample species, RBC and WBC were mixed at equal concentrations of 5 million cells/mL and separated according to their different levitation characteristics as illustrated in FIG.
2A. The same samples were also imaged using regular microscopy to confirm the types of cells that levitated different heights as illustrated in FIGS. 2B-2D. Overlapped images of WBC labeled with CD45 and the bright field image of the mixed RBC and WBC sample clearly validated the i-LEV results as illustrated in FIGS. 2A-2D. Additionally, live-dead assays were performed with WBC and RBC. First, WBC were stained and frozen overnight.
These dead cells were then spiked into RBC samples at equal concentrations. The i-LEV
system shows only RBC levitated at the middle of the channel, whereas dead WBC aggregated at the base as illustrated in FIG. 2E. To validate the results, the dead WBC was stained with DAPI and visualized them by fluorescence microscopy. The overlapped bright field image of WBC
seen in FIG. 2F and DAPI-stained WBC seen in FIG. 2G confirmed the dead-live assay results as illustrated in FIGS. 2F-2H.
concentration in this example experiment is around 30 mM, as this concentration allows optimal levitation while keeping the cells at an adequate distance from the capillary walls.
In this condition, the resulting bands are easy to distinguish.
blood were performed to test the stabilization time at different concentrations. The exponential time constants for the stabilization curves were 5.8 min for 450 million cells/mL and 3.3 min for 90 million cells/mL. As further shown in FIG. 3B, the blood cell concentration versus time curves show that the equilibration time for the curves was again 15 min. Higher concentrations of blood cells took longer to equilibrate.
Further validation experiments were performed to assess the changes in blood bandwidth at different concentrations. RBC were imaged with the i-LEV system at concentrations of 250, 125, 63, 50, 25 and 0.8 million cells/mL. Each sample was quantified using a hemocytometer to confirm the calculated blood counts. To assess the cell concentrations, the width of the levitated blood band across the channel was measured by dividing the total area of the blood by width of the illuminated region. At higher concentrations (between 50 to 250 million cells/mL), the blood width versus concentrations curves were linear with a slope of 0.6 micrometers per million cells/mL. However, as the cell concentration decreased (for example, from 0.8 to 25 million cells/mL) the curves lost their linearity as can be seen in FIG. 3D. For blood cell concentrations above 50 million cells/mL, it was observed that the width of the blood band during levitation was correlated with the cell concentration. WBC
were also imaged at varying concentrations ranging from 1 to 5 million cells/mL and plotted the concentration against the width of the blood band as illustrated in FIGS. 3E-3F.
WBC concentrations also correlated with the width of the blood band in a linear manner.
After diluting the RBC concentration to 100,000 cells/mL or lower, individual cells in the illuminated area were quantified using simple image processing tools as illustrated in FIGS.
4A-4B. Finally, polyethylene beads were levitated in the capillaries to check the levitation resolution of the platform and show its potential to calculate densities for different samples and cells. Beads with various sizes between 10-100 um in diameter with densities of 1.025 g mL-1, 1.031 g mL-1, 1.044 g mL-1 or 1.064 g mL-1 showed distinct levitation heights in 30 mM Gd+ as illustrated in FIG. 4C. It was also observed that beads with 1.064 g mL-1 density had different levitation heights in different Gd+ concentrations (10 mM, 30 mM, 60 mM) as illustrated in FIG. 4D.
system reliably analyses blood cell counts and can also detect individual cells. It is a rapid, portable, easy to use and affordable platform that leverages the availability of smartphones to address a medical need and count RBC as well as WBC from unprocessed whole blood.
In the state of the art at present, blood processing is a clinical procedure and requires extensive materials and equipment, as well as trained professionals.
Therefore, it can currently not be implemented in the POC setting. The disclosed system could, however, enable blood analyses from home and facilitate disease diagnosis and monitoring.
system once again particularly relevant for developing countries.
could be extended variety applications including (1) monitoring the effect of drugs, metabolites, chemicals; (2) studying the mechanobiology of cells under different conditions; and (3) studying embryo and oocyte differentiation and accessing the health of embryos/oocytes.
Example II: Monitoring the effect of drugs using levitation device
7B), chloroquine concentrations of 1.25, 2.5, 5 and 7.5 mg/mL are spiked into blood and levitation heights of these images were analyzed. FIG. 7D illustrates the actual images that were analyzed to produce the plots of FIG. 7B and 7C. In FIG. 7D, a certain density beads was added each time for calibration experiments.
of chloroquine concentration. FIG. 6B is then a graph showing the area analysis.
Area of the blood were measured using an image) script developed in the lab. Area shows the viability of the cells. As the cells apoptosis with time, area gets smaller. Looking at FIG. 6C, the levitation height is measured using same script. Height is directly correlated with density of cells. Higher drug concentration leads to higher density of blood within 50 minutes.
Example III: Mechanobiology studies using the levitation system
provided in FIG. 9C), density plots provide more details and enable saddle differences to be seen.
Thus, the i-LEV device could be used for cell mechanics studies, including aging and stem cell differentiation.
Example IV: Embryo differentiation studies and assessing the health of embryos
Accordingly, this system could be applied to monitor and understand healthy embryos which are levitated at different height.
Example V: Updated rendering of i-LEV design
High Throughput Magnetic Levitation Cell Separation
is separately infused through top and middle inlets.
Example VI: Density separation of test beads
Example VII: Density separation of breast cancer cells
Example VIII: Alternative two-magnet design
The first microfluidic design incorporates a long, straight channel that is split into two (top and bottom channels) by a thin film near the outlet, each of which is connected to an outlet, respectively. Two permanent NdFeB magnets are respectively placed on the top and bottom of the channel, with the same poles facing each other. Such a design for a magnetic levitation-based high-throughput blood cells sorting device can be found in FIG. 18, for example, which includes a pair of magnets.
Example IX: Plasma Blood Analysis
Whereas, blood cell sorting was not found at higher flow rate such as 30 and 50 uL/min, since blood cells was also recovered at top channel. Due to the insufficient duration at higher flow rate, blood cells are not levitated and separated into homogeneous layer that cells are flowed on both channels. Thus, 20uL/min is chosen as optimal flow rate for blood cell sorting in the exemplary system the tests were performed on. In FIG. 19D, total protein analysis of plasma collected from top channel and compared with plasma, which is separated by the centrifugation and it was found that the concentration of protein on plasma form the top channel is recovered as found in the traditional centrifugation method.
It is concluded that under the optimized condition, the developed high throughput MagLev-based devices (i.e., those sorting devices previous described) is efficiently sorting the blood cells.
Example X: Fluorescence-activated cell sorting (FACS) analysis
analysis.
As shown in FIG. 20A, both RBCs and WBCs were observed in whole blood and, as shown in FIG. 20B, blood cells separated from bottom channel of the Maglev device, whereas at top channel of the device, blood cells were not observed as shown in FIG. 20C. The obtained results further conforms that under optimized conditions the developed Maglev device is able to separate the blood cells and plasma from whole blood.
Example XI: RBCs and WBCs separation from whole blood
21B. FIG. 21C also indicates that about 70% of WBCs were collected from the top channel, which may due that most of the WBCs are levitated higher than 100 pm height that flows through the top channel. The obtained results proved that the developed high throughput MagLey device is able to isolate rare cell, such as CTCs and fetal red blood cells from whole blood and tumors.
Example XII: Sorting cancer cells in blood
For example, cancer cells can be easily separated from a complex mixture (i.e., blood) due to their large intrinsic density difference. In the disclosed microfluidic design, CTC/CTM
that are typically levitated above blood cells can be collected from the top channel.
We first tested the platform for plasma separation, in which whole blood sample with 30 mM
Gd3+ is flowed through the channel at a constant flow rate, 20 4/min. Under the magnetic field, cells are moving from high magnetic induction to low, and levitated in a specific height in the channel according to their density signature. Due to the high density, blood cells RBCs and WBCs were flowed below the divider and collected at bottom outlet, while plasma flows above and collected from top channel outlet. Similarly, by reducing the bottom channel height into 100 m, WBCs were separated and collected from the top channel, which shows the potential of this device that can also separate the CTCs from the whole blood sample.
Example XIII: Cell separation / blocking using a magnetic field
Two permanent neodymium magnets are placed on top and bottom of the channel as same pole facing to each other. For the plasma separation experiment, a paramagnetic medium gadolinium (Gd3+) spiked blood sample is flowed in to inlet using a syringe pump at a constant flow rate. Due to high magnetic induction at near the magnetic edge, cells are blocked, and stacked back in inlet chamber and allows only plasma into channel that was collected at outlet.
23B). FIGS. 24 and 25 shows the optimization of paramagnetic medium Gd3+ concentration for efficient plasma separation from blood sample. In FIG. 25A, the blood is shown being loaded into the cell blocking based plasma separation device (i.e., the MagLev), in FIG.
24 detailed views are shown illustrating the blocking, and FIG. 25B shows the plasma collection at outlet (from 100 mM Gd3+ spiked 10 time diluted with PBS blood sample flowed at constant flow rate, 20 L/min according to the experiment described below).
Gd3+.
Multi-cellular 3D assembly based on magnetic signatures and simulation of microgravity
cell assembly with controlled shape. This method could surpass conventional 21) tissue culture by offering cell culturing conditions able to mimic the in vivo cell-cell and cell-ECM
interactions and organization. Compared to other methods, the magnetic levitation methodologies described herein do not require engineered scaffolds or magnetic nanoparticles (for levitation). Assembled cell aggregates can be used for tissue engineering, disease modeling, drug-screening and cell/tissue biology study.
Example XIV: Spheroid assembly method
Example XV: Second example of multi-cellular 3D assembly based on magnetic signatures
shows co-levitation of polymeric strand and GFP-HUVECs cells after 10 min and the image in FIG. 27F
shows the same after 1 day in 75 mM Gadolinium concentration. In all of these images, the scale bar is 200 um.
Example XVI: Method for cell sorting, recovery and characterization based on magnetic levitation
Example XVII: Spheroid aggregation time study
Magnetic levitation (LEV) and hanging drop (HD) were compared. After 8 hours, stable cell aggregates were formed in the magnetic levitation device. The white scale bar in these images is 200 um.
Example XVIII: Effects of Gd concentration and levitation on cell viability
Spheroid viability was then assessed with live/dead assay (calcein/ethidium homodimer-1). The obtained results show that the majority of the cells in the aggregates are alive. The white scale bar is 100 um.
Example XIX: Merging of spheroids
Bright-field and fluorescence images are presented.
Example XX: Functionality study of 3T3 spheroids
Gd). The spheroids were then placed in non-adhesive 96-well plate, embedded within fibrin gel (20mg/m1) and cultured for 5 days as illustrated in FIG. 32A. Every day bright-field images of the spheroids were collected. In FIG. 32A, the scale bar is 200 um for days 0-2 and 500 um for days 3-5. FIG. 32B illustrates the spheroids immunostaining after levitation and FIG. 32C illustrates after 5 days in fibrin gel. Nuclei (DAPI, blue), cell proliferation (ki67, magenta), collagen I (red) secretion and actin filaments (green) [colors depicted in greyscale]. The obtained results show that the assembled spheroids are functional. The white scale bar is 200 um.
Example XXI: Microfluidic magnetic levitation system for assembly
Therefore, multiple spheroids (one for each compartment) can be obtained in a high-throughput fashion. FIG. 33B depicts one such spheroid formed at a bend. Cell concentrations of 150 thousand cells/ml were used (48 hours, 60mM Gd+). In FIG. 33B the scale bar is 200 mm.
Assorted additional applications of these magnetic levitation systems and tools
Example XXII: Magnetic bead strategy for target cell (i.e. bacteria, yeast, virus, pathogen, circulating tumor cells, circulating epithelial cells, etc.) identification and enrichment with magnetic levitation
After capture of a specific pathogen type with the magnetic particles, its magnetic signature will increase which will cause the target cell/molecule/pathogen to sink toward to bottom of the microchannels. Captured cells/molecules then can be flushed out and enriched for further analysis, (i.e., antibiotic susceptibility analysis).
Example XXIII: High throughput bacterial cell isolation and antibiotic susceptibility testing from clinical samples
After sample injection into the high throughput microfluidic levitation system, bacteria and blood cells will be levitated according to their density signatures and separated into homogenous layers. Due to their higher density, bacterial cells will levitate at a different height and will be collected at another (i.e., bottom) outlet, while red and white blood cells will levitate at a different height and be collected from the other outlets.
The high throughput microfluidic platform also enables a unique ability to reuse the isolated pathogens and perform repeated antibiotic susceptibility tests on the same samples. With current clinical assays, bacteria need to be cultured and expanded in number given that the number of isolated bacterial cells is about 10-100 cells/ml. In the microfluidic levitation platform, the bacterial cells will be tested with one antibiotic and investigated for rapid changes in the levitation profiles. If the bacterial population is resistant to the treatment, the bacterial cells will be flowed out of the channels, the chambers will be washed with PBS
to get rid of the remaining antibiotic solution and the same bacterial population will be tested with another antibiotic candidate. Thus, this capability will significantly eliminate the need to culture the pathogens that are very low in number for future antibiotic susceptibility testing.
Example XXIV: Levitation platform applied to profile the blood cells of patients suffering from chronic diseases
from healthy controls.
Example XXV: Continuous monitoring and levitation profiling of red blood cells from patients suffering from chronic diseases
Example XXVI: Screening and monitoring the effect of metabolites, chemicals using levitation device
37B illustrates cell viability, number of live and dead white blood cells can be monitored, detected and counted according to their levitation profiles in real-time with single-cell resolution.
Example XXVII: Antimicrobial applications
Example XXVIII: Levitation of bacterial cells
Overnight cultures of E. coli (Dh5a) was diluted in FBS and levitated in 100 mM Gd+
solution. Bacteria cells formed their intrinsic levitation bands after one hour of levitation.
Example XXIX: Rapid detection of bacterial cells from surfaces
and 30 ul swap sample was levitated in 200 mM Gd+ solution. Bacteria cells formed their intrinsic levitation bands within 30 minutes. Thus, bacteria can be rapidly detected from surfaces with the portable levitation system.
Example XXX: Levitation of other types of moieties and cells
Example XXXI: Prediction of oocyte quality using magnetic levitation
Example XXXII: Levitation profiles of uninfected vs. HIV-infected CD4 T cells
paramagnetic solution, respectively. The samples were levitated for 20 minutes and the levitation profiles were compared. As illustrated in FIG. 44A and 44B, uninfected and HIV-infected blood CD4 T cells have significantly different levitation profiles in the magnetic levitation-based platform.
solution with 30 mM paramagnetic solution, respectively, as shown in FIG. 45A (and quantified in FIG.
458). HIV-infected blood CD4 T cells were used as controls. The samples were levitated for 20 minutes and the levitation profiles were compared. It was shown that stimulation with anti-CD3/CD28/IL2 beads and anti-CD3/CD28/IL2 antibody changes the levitation profiles of HIV-infected blood CD4 T cells.
Example XXXIII: Additional design for microfluidic high-throughput magnetic levitation platform (i.e., the addition of needles and suction)
In addition, these needles can be located anywhere in the 3D spatial domain of the channel or outside. Samples (i.e., beads, cancer cells, blood, etc.) can be mixed in the inlet and then a variety of negative pressure and flow rates can be used to withdraw the samples according to their densities. Samples with lower density or lower magnetic susceptibility can be withdrawn by a microfluidic pump with the bottom needle and collected in the bottom outlet tube. Samples with higher density or higher magnetic susceptibility can be withdrawn with the bottom needle and collected in the bottom outlet tube.
Other design parameters of the magnetic platform (e.g., magnet strength, geometric positioning of magnets, dimensions) can further be modified accordingly to enable rapid processing and better sorting.
SUCtiOrlbottom needle).
Example XXXIV: Results for droplet sorting and droplet sequencing
Droplets encapsulating different types of cells can have different levitation profiles and then sorted and collected for further characterization for genomic, transcriptomic, proteomic, and metabolic analysis. Droplets containing live and dead cells can have different levitation profiles. This can be used for Drop-Seq applications to separate droplets containing dead cells vs. live cells. Droplets encapsulating only the live cells then can be used for Drop-Seq. This capability will significantly reduce the cost and processing time for Droplet-Sequencing methods.
Example XXXV: Collection of data for cancer studies
The levitation chip can separate circulating tumor cells and circulating tumor clusters within the same device at the same time. As illustrated in FIG. 51A, a procedure is depicted that can be used to test for the presence of calcein-labeled single cells (CTC) and aggregates of cancer cells (CTM). Single cells of kidney cancer cells (CTC) and clusters (CTM) of kidney cancer cells can be separated and sorted at the same time, within the same device be detected without using any labels, in spike-in experiments with WBCs. Then, in FIG. 51B, the separation efficiency of cancer cells from blood via magnetic blueprinting is shown.
Efficiency at different concentrations of spiked cancer cells (100 cells/mL, 1,000 cells/mL, 100,000 cells/mL) were analyzed and compared for different cancer cell types (i.e., lung, breast and kidney cancer). Separation efficiency is defined as the ratio of cancer cells levitated above the blood cell band to the total number of spiked cancer cells. (N=3 independent experiments, data are represented as the mean standard error of the mean (SEM)).
and then levitated in 30 mM Gd+ solution in the magnetic levitation device..
After 20 minutes of levitation, CTCs and clusters of CTCs (blue circles, i.e., the three circles on the right) were levitated above the band consisting of RBCs and WBCs depicted in FIG. 51A, rightmost image. These putative CTCs were observed to be very light in density, as they have levitated at the top of the microcapillary channels.
Claims (45)
a levitation device for separating a heterogeneous population of moieties wherein variances in the moieties are based on differences in magnetic susceptibility and/or intrinsic density, the device including at least one magnet producing a magnetic field, wherein the magnetic field is sized to interact with a microcapillary or microfluidic channel for reception of a sample containing the heterogeneous population of moieties;
wherein the microcapillary or microfluidic channel is defined by a plurality of layers having portions of the microcapillary or microfluidic channel formed therein, at least one of the plurality of layers providing an inlet channel into the microcapillary or microfluidic channel and at least two of the plurality of layers providing separate outlets from the microcapillary or microfluidic channel.
flowing a sample including the heterogeneous population of moieties and a paramagnetic medium into the at least one inlet and the microcapillary or microfluidic channel;
applying a magnetic field to the sample including the heterogeneous population of moieties to separate the heterogeneous population based on a difference in at least one of magnetic susceptibility and intrinsic density between individual members of the heterogeneous population of moieties and the paramagnetic medium;
thereafter flowing a first portion of the sample out of a top outlet and a second portion of the sample out of a bottom outlet, thereby separating a first group of the heterogeneous population of moieties from a second group of the heterogeneous population of moieties.
flowing a sample including the population of moieties and a paramagnetic medium from an inlet into the microcapillary or microfluidic channel towards an outlet; and while the sample is in the microcapillary or microfluidic channel, applying a magnetic field using the at least one magnet to the sample, wherein the application of the magnetic field blocks at least some, but not all, of members of the population of moieties from flowing towards the outlet, thereby separating the population of moieties.
introducing a sample including the population of moieties and a paramagnetic medium into the microcapillary or microfluidic channel; and while the sample is in the microcapillary or microfluidic channel, applying a magnetic field using the at least one magnet to the sample, wherein the application of the magnetic field levitates at least a portion of the population of moieties to place the population of moieties in a spatial relationship to one another in which the population of moieties aggregate to form the multi-moiety assembly.
a levitation device for separating a heterogeneous population of cells, wherein the variances in the cells are based on differences in magnetic susceptibility, the device including at least one magnet producing a magnetic field, wherein the at least one magnet is proximate a microcapillary or microfluidic channel for reception of a sample containing a heterogeneous population of cells;
a light source; and a frame, the frame supporting the levitation device, and the light source, and is further configured to support an imaging device, the frame supporting the light source in a position to transmit light through the levitation device to the imaging device for real-time observation of at least a portion of heterogeneous population of cells.
placing the sample of the population of cells in a microcapillary or microfluidic channel of a levitation device;
placing an imaging device in a frame;
separating the population of cells in the levitation device; and while separating the population of cells in the levitation device, using the imaging device to collect images of the separation of the population of cells.
38. The method of claim 34, further comprising at least one of counting and quantifying at least some of the population of cells from the collected images of the population of cells.
a levitation device for separating a heterogeneous population of moieties wherein variances in the moieties are based on differences in magnetic susceptibility and/or intrinsic density, the device including at least one magnet producing a magnetic field, wherein the magnetic field is sized to interact with a microcapillary or microfluidic channel for reception of a sample containing the heterogeneous population of moieties;
at least one needle at the inlet and/or outlet of the microcapillary or microfluidic channel introducing or withdrawing fluid from the microcapillary or microfluidic channel at a respective pre-defined position over the height and/or width of the microcapillary or microfluidic channel.
39. The magnetic levitation-based system of claim 38, comprising a plurality of needles at the inlet or the outlet, each of the plurality of needles being in communication with the microcapillary or microfluidic channel at a different spatial position.
40. The magnetic levitation-based system of claim 38, wherein a first injection needle at the inlet is at a different height than a second suction needle at the outlet.
41. A method of evaluating a quality of individual embryos and oocytes for use in reproductive medicine, the method comprising:
placing a sample including one or more embryos or oocytes in a microcapillary or microfluidic channel of a levitation device;
levitating the sample including embryos or oocytes in the levitation device;
and grading the quality of the embryos or oocytes on one or more of the density and levitation profile.
42. The method of claim 41, further comprising the step of selecting one or more of the embryos or oocytes based on the grading of the quality of the embryos or oocytes on one or more of the density and levitation profile and employing the one or more embryos or oocytes in an in vitro fertilization procedure.
43. A method for levitating a plurality of moieties encapsulated in droplets in a magnetic levitation system for droplet sequencing, the method comprising the steps of:
encapsulating the plurality of moieties in a plurality of droplets and suspending the plurality of droplets in a sample;
placing the sample containing the plurality of droplets in a microcapillary or microfluidic channel of the magnetic levitation system; and levitating the plurality of droplets in the magnetic levitation system.
44. The method of claim 44, wherein the sample further includes a plurality of droplets not encapsulating any of the plurality of moieties.
a levitation device for separating a heterogeneous population of moieties wherein variances in the moieties are based on differences in magnetic susceptibility and/or intrinsic density, the device including at least one magnet producing a magnetic field, wherein the magnetic field is sized to interact with a microcapillary or microfluidic channel for reception of a sample containing the heterogeneous population of moieties;
at least one needle at the inlet and/or outlet of the microcapillary or microfluidic channel introducing or withdrawing fluid from the microcapillary or microfluidic channel at a respective pre-defined position over the height and/or width of the microcapillary or microfluidic channel.
placing a sample including one or more embryos or oocytes in a microcapillary or microfluidic channel of a levitation device;
levitating the sample including embryos or oocytes in the levitation device;
and grading the quality of the embryos or oocytes on one or more of the density and levitation profile.
encapsulating the plurality of moieties in a plurality of droplets and suspending the plurality of droplets in a sample;
placing the sample containing the plurality of droplets in a microcapillary or microfluidic channel of the magnetic levitation system; and levitating the plurality of droplets in the magnetic levitation system.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US201562236692P | 2015-10-02 | 2015-10-02 | |
US62/236,692 | 2015-10-02 | ||
PCT/US2016/054987 WO2017059353A1 (en) | 2015-10-02 | 2016-09-30 | Sorting biological and non-biological moieties using magnetic levitation |
Publications (1)
Publication Number | Publication Date |
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TR201910569A2 (en) * | 2019-07-16 | 2021-02-22 | Izmir Yueksek Teknoloji Enstituesue | DESIGN AND PRODUCTION OF PARTIAL GRAVITY PLATFORM USING MAGNETIC FORCES FOR CELLULAR APPLICATIONS |
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CN112326503B (en) * | 2020-10-14 | 2022-03-08 | 上海交通大学 | Halbach array type antimagnetic suspension cell density detection and separation device and method |
WO2022086915A1 (en) * | 2020-10-20 | 2022-04-28 | The Board Of Trustees Of The Leland Stanford Junior University | Isolation of different extracellular vesicle (ev) subpopulations |
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WO2022251582A1 (en) * | 2021-05-27 | 2022-12-01 | The Board Of Trustees Of The Leland Stanford Junior University | Densitometry-based sorting for embryo health classification |
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KR20180061336A (en) | 2018-06-07 |
JP2023081880A (en) | 2023-06-13 |
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